Description: Hematopoietic stem cells are a potent therapeutic tool capable of fully reconstituting immunologic function in a transplanted host. Genetic manipulation of stem cells represents a means of generating immune function resistant to HIV infection and thereby the potential of immune restitution beyond that possible with HAART alone. Limiting the practical application of stem cell based therapeutics is the low efficiency of engraftment imposing unrealistic quantitative demands on isolation and transduction protocols. The ability to expand stem cells ex vivo would overcome major limitations of the field, but current protocols involve cytokine stimulation that generally sacrifice multipotentiality for increased cell numbers. This application proposes a model of stem cell proliferation regulated by dominant inhibitory signals and presents data to support the model using mice engineered to be deficient in the cyclin dependent kinase inhibitor (CDKI), p21. The aims of this application are to: 1). Further define proximate mediators of cell cycle inhibition in stem cells focusing on a) the p21 family member, p27, using gene targeted mice and b) the interrelationship of the inhibitory TGF-b signaling pathway with p21 and p27. 2). Alter cell cycle inhibitory control as a means of inducing cell proliferation, testing whether cells entering cell cycle by this mechanism avoid the pro-differentiative effect of cytokines. The potential for unlinking proliferation and differentiation to achieve ex vivo expansion of stem cells will be defined. 3). Determine if HIV-1 exposure alters the expression of cell cycle inhibitors in stem cells or affects quiescent stem cell responsiveness to CDKI down-modulation. The potential of manipulating stem cells by targeting cell cycle inhibitors as a means of expanding stem cells in the specific context of HIV disease will be defined. Accomplishment of these aims will guide specific strategies for stem cell gene therapy of HIV disease based on principles of stem cell regulation.